The long term objective of this research is to investigate the genetics and molecular biology of a virulence-associated property, plasmid-mediated iron uptake, which plays an important role in the pathogenesis of many invasive septicemic diseases of man and domestic animals. We are presently using as a model system the fish pathogen Vibrio anguillarum which causes a terminal hemorrhagic septicemia. The features of the plasmid-mediated virulence determinant makes it a very attactive model to study host bacterial interacions. We have used transposition mutagenesis, molecular cloning and DNA sequencing to define at least four components of the pJM1 plasmid-mediated iron uptake system: a siderophore; a receptor for iron-siderophore complexes which is the 86 kd outer membrane protein OM2; a regulatory trans-acting factor and RSV1 repeated sequences that flank the iron uptake region in the pJM1 plasmid. The specific roles that each of these four components play in the regulation of the iron uptake process and virulence in V. anguillarum will be elucidated by the proposed investigation. The specific aims to achieve these objectives are: 1. Isolation and structure determination of the V. anguillarum siderophore. 2. Complementation analysis of cloned pJM1 iron uptake regions. 3. Characterization of the cloned OM2 protein gene. 4. Cloning and characterization of the trans-acting regulatory factor that enhances the production of siderophore activity. 5. Investigation of the nature of the increased siderophore production and high iron uptake proficiency of a V. anguillarum isolate and its relationship to the tandem duplication of one of the two repeated sequences flanking the pJM1 iron uptake region. Repeated sequences identified as IS1-like elements, flank the iron uptake region of another virulence associated plasmid, pCo1V-K30, found in human invasive strins of Escherichia coli. The iron uptake genes within the region flanked by the repeated sequences appeared to be very conserved in both the pCo1V-K30 and the pJM1 plasmids, as well as in other plasmids and chromosomes. The existence of the repeated sequences bracketing these virulence determinants may have contributed to their recombinational mobility. Thus, this project will not only elucidate the role of iron uptake in the pathogenesis of invasive bacterial disease, but will also contribute to the understanding of the mechanisms by which these virulence factors have spread epidemiologically in nature.